S-2-氯丙酸脱卤酶在毕赤酵母中的重组表达

根据S-2-氯丙酸脱卤酶的基因序列设计引物,克隆重组大肠杆菌中的S-2-氯丙酸脱卤酶基因。将目的基因片段构建到pPIC9K载体,经Sac I单酶切后转化到毕赤酵母GS115中,通过G418抗性YPD平板筛选高拷贝重组子,甲醇诱导成功实现胞外活性表达。重点考察了基因拷贝数和甲醇浓度的影响,结果表明重组子含有12个拷贝,甲醇浓度为1%时较佳,重组脱卤酶的酶活可达236U/L。重组毕赤酵母具有很好的遗传稳定性。对该酶最适反应温度及pH进行研究,表明其最适反应温度为50℃,最适pH为9.5。     

幽门螺杆菌BabA蛋白N段的基因克隆、表达纯化及体外粘附活性评价

目的:克隆幽门螺杆菌BabA蛋白的N段(氨基酸1-437位)基因(BabA1),构建其原核表达质粒,表达并纯化获得携带6×His标签的BabA1融合蛋白,评价融合蛋白的体外粘附活性。方法:以幽门螺杆菌J99菌株基因组DNA为模板,通过PCR扩增获得BabA1基因片段,并定向克隆至原核表达载体pET-32a(+)。经酶切及测序分析正确后,将重组质粒pET32a-BabA1转化大肠杆菌BL21(DE3)菌株,进行IPTG诱导表达,并对表达产物进行镍柱纯化、SDS-PAGE和Westernblot鉴定。采用菌落计数法评价BabA1融合蛋白在体外的细胞粘附活性。结果:成功扩增了BabA1基因,构建了pET32a-BabA1原核表达质粒,并通过优化该表达系统的最适诱导和纯化条件,获得了具有活性的6×His-BabA1融合蛋白,后者能显著增强大肠杆菌BL21(DE3)在体外对胃癌MFC细胞的粘附。结论:成功诱导表达并纯化获得了有粘附活性的6×His-BabA1融合蛋白,为进一步研究其免疫活性和生物学功能奠定了基础。     

重组蛋白TAT-NLS-Nkx6.2在大肠杆菌的表达纯化及活性测定

目的:构建重组基因TAT-NLS-Nkx6.2的原核表达载体,获得重组TN-Nkx6.2蛋白,并验证其生物学活性。方法:该实验先将人类免疫缺陷病毒HIV-1的反式激活因子TAT、核定位信号NLS、Nkx6.2基因片段连接合成目的基因TAT-NLS-Nkx6.2。之后构建重组质粒pET-28a-TNNkx6.2并将其转化入E.coli DH5α中克隆,再转化至E.coli BL21(DE3)中用IPTG诱导表达,经组氨酸Ni2+亲和层析纯化融合蛋白,并用SDS-PAGE及Western blot鉴定融合蛋白,荧光免疫组化染色观察TN-Nkx6.2在小鼠脑组织分布。结果:成功表达并获得纯度90%以上的TN-Nkx6.2蛋白,经Western Blot鉴定,TN-Nkx6.2有良好的免疫原性和免疫反应性荧光免疫组化染色证明TNNkx6.2与小鼠大脑细胞结合并内化进入胞浆及胞核。结论:获得的融合蛋白TN-Nkx6.2具有生物学活性,并能穿过血脑屏障与小鼠大脑细胞结合,并内化进入胞核和胞浆,为深入探讨同源盒基因Nkx6.2在甲状腺减低模型的分子机制研究奠定了物质基础。     

人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析

利用毕赤酵母(Pichia pastoris)重组表达人源类溶菌酶蛋白6(human lysozyme-like protein6,h Lyzl6),对其酶学性质进行分析。根据毕赤酵母密码子偏爱性设计并人工合成h Lyzl6基因,将其连接至含有乙醇氧化酶启动子(AOX1)的p PIC9K质粒构建重组表达载体p PIC9K-hlyzl6;重组表达载体经线性化后电转化入毕赤酵母GS115感受态细胞,经G418筛选获得高拷贝重组菌株后进行甲醇诱导表达。经甲醇诱导72 h后发酵液上清中酶活性达到最高值,发酵液上清经SDSPAGE检测在14.8 k Da处有重组h Lyzl6蛋白条带,分子量符合预期,通过甲壳素亲和层析可对其进行纯化;采用比浊法测定h Lyzl6酶学活性,结果表明h Lyzl6对溶壁微球菌(Micrococcus lysodeikticus)有较好的杀灭作用,最适反应温度为40℃,最适p H为5.5,其酶活力为54 700U/mg,Cu2+对其活性有明显抑制,EC50为30.2799 mg/L。采用基因工程方法首次在毕赤酵母GS115成功表达了重组h Lyzl6,证实其在体外具有杀菌活性,初步揭示h Lyzl6在男性生殖系统先天性免疫中发挥了一定作用,为进一步研究h Lyzl6的功能和应用开发奠定了基础。     

毕赤酵母STE13基因敲除对GGH表达及其生物活性的影响

目的:重组毕赤酵母GS115/pPIC9K-GGH表达的人胰高血糖素样肽-1-人血清白蛋白融合蛋白[(GLP-1 A2G)2-HSA,GGH]的细胞活性测评结果显示生物活性较低,这可能与其表达过程中蛋白降解有一定关联。通过对毕赤酵母GS115 STE13基因的敲除,获得一株完整表达蛋白GGH的毕赤酵母宿主,并进一步提高其表达产物GGH的生物活性。方法:采用基于PCR技术介导的Cre-Loxp系统重组技术成功敲除宿主菌GS115的STE13基因,得到一株STE13缺陷型菌株GS115/D13,并通过细胞活性测评系统检测GS115/D13表达产物GGH的生物活性。结果:通过对STE13基因缺陷型菌株GS115/D13与出发菌株GS115/W发酵表达对比,显示GS115/D13菌株表达融合蛋白GGH生物活性有明显提升,且表达量提高了25.8%,两菌株生长无明显差异。结论:STE13基因的敲除成功缓解了GGH表达过程中的降解问题,并相对出发菌株GS115/W大幅度提高了蛋白生物活性。     

利用酿酒酵母稳定完整表达融合蛋白hPTH(1-34)_(ab)-HSA

为稳定完整表达人甲状旁腺激素(hPTH)(1-34)二联体与人血清白蛋白(HSA)融合蛋白,以酿酒酵母为宿主,利用重叠PCR技术,将α-factor基因拼接到hPTH(1-34)ab-HSA基因片段上,获得α-hPTH(1-34)ab-HSA,并插入穿梭表达质粒pYX212,构建了基因工程菌S.cerevisiae W303-1A/pYX-α-hPTH(1-34)ab-HSA。经Western blot分析及N端氨基酸测序表明,该工程菌表达分泌了具有HSA和hPTH抗原性的完整hPTH(1-34)ab-HSA融合蛋白,解决了毕赤酵母不能稳定完整表达融合蛋白hPTH(1-34)ab-HSA的问题。     

栖热菌噬菌体TSP4 dCTP脱氨酶基因的克隆表达

将编码栖热菌噬菌体TSP4菌株的dCTP脱氨酶tspdCD基因亚克隆到表达载体pET-32a中,并将重组质粒pET-32a-tspdCD转化至大肠杆菌Rosetta(DE3)中进行诱导表达。SDS-PAGE分析结果显示,目的蛋白经IPTG诱导后在大肠杆菌Rosetta(DE3)中以可溶性形式高效表达。通过Ni-NTA agarose亲和层析柱纯化表达的tspdCD,并对其活性,最适作用温度、pH、底物特异性以及金属离子和有机溶剂对其活性的影响进行测定,检测结果表明重组蛋白活性达到4.12U/mg,它的最适作用温度和pH分别为60℃和7.5,最佳反应底物是dCTP,2mmol/L的Ca2+和Mg2+对其活性具有明显的促进作用,而同浓度的Ni2+和Cu2+对其活性产生了明显的抑制作用,10%(V/V)的乙酸乙酯和异丙醇对其活性有很明显的促进作用,而同体积比的丙酮对其活性产生明显的抑制作用。实现了tspdCD在大肠杆菌系统中的功能性表达,为进一步研究高温噬菌体tspdCD的功能奠定了基础。     

皱纹盘鲍HdhTPX2基因在毕赤酵母中的表达及抗氧化活性研究

为研究皱纹盘鲍Haliotis discus hannai Ino硫氧还蛋白过氧化物酶(TPX2)蛋白的抗氧化活性,将HdhTPX2基因克隆至pPIC9K载体,通过电击转化至毕赤酵母GS115菌株中,获得重组表达质粒pPIC9K-HdhTPX2,经甲醇诱导及亲和层析纯化得到重组HdhTPX2蛋白,并进行质谱鉴定及体外抗氧化功能检测。结果表明:本研究中成功构建了重组毕赤酵母菌株GS115/pPIC9K-HdhTPX2,经表达条件的优化,在pH为7的培养基中用0.5%甲醇诱导表达72 h,分泌表达上清液中得到相对分子质量约为25 000的稳定表达产物,纯化后的蛋白经质谱鉴定为目的蛋白;体外活性测定发现,该蛋白清除羟自由基(·OH)能力强于维生素C,并对H_2O_2引起的细胞损伤具有一定的保护作用。研究表明,皱纹盘鲍硫氧还蛋白过氧化物酶HdhTPX2在毕赤酵母表达系统中得到高效表达,重组表达产物具有抗氧化活性功能。     

C11orf17蛋白的原核表达及功能预测

目的利用基因工程技术获取原核表达的C11orf17蛋白,结合生物信息学初步预测其功能。方法以K562细胞的cDNA为模板,PCR扩增C11orf17的基因序列;双酶切后将目的基因亚克隆至原核表达载体pET-32a上,将测序正确的重组表达质粒pET-32a-C11orf17转化到E.coli BL21,经诱导后SDS-PAGE及Western blot检测融合蛋白表达,并分析其可溶性。采用UGET软件预测C11orf17共表达基因,蛋白质互作数据库挖掘C11orf17基因已知互作蛋白,在线软件Gather富集共表达基因生物功能。结果成功构建重组表达质粒pET-32aC11orf17,此重组体经诱导后能在E.coli BL21高效表达C11orf17的融合蛋白,UGET分析C11orf17前300共表达探针中,包含267个已知基因,共表达基因主要富集细胞交流、信号传导、细胞周期基因功能本体。蛋白互作数据库表明,C11orf17与PRKACA蛋白互作,后者为细胞周期调控重要分子。结论在原核细胞中成功表达出C11orf17蛋白,生物信息预测其可能是细胞周期调控重要新分子,为后续研究提供了方向和线索。     

皱纹盘鲍HdhTPX2基因在毕赤酵母中的表达及抗氧化活性研究

为研究皱纹盘鲍Haliotis discus hannai Ino硫氧还蛋白过氧化物酶(TPX2)蛋白的抗氧化活性,将HdhTPX2基因克隆至pPIC9K载体,通过电击转化至毕赤酵母GS115菌株中,获得重组表达质粒pPIC9K-HdhTPX2,经甲醇诱导及亲和层析纯化得到重组HdhTPX2蛋白,并进行质谱鉴定及体外抗氧化功能检测。结果表明:本研究中成功构建了重组毕赤酵母菌株GS115/pPIC9K-HdhTPX2,经表达条件的优化,在pH为7的培养基中用0.5%甲醇诱导表达72 h,分泌表达上清液中得到相对分子质量约为25 000的稳定表达产物,纯化后的蛋白经质谱鉴定为目的蛋白;体外活性测定发现,该蛋白清除羟自由基(·OH)能力强于维生素C,并对H_2O_2引起的细胞损伤具有一定的保护作用。研究表明,皱纹盘鲍硫氧还蛋白过氧化物酶HdhTPX2在毕赤酵母表达系统中得到高效表达,重组表达产物具有抗氧化活性功能。     

粉尘螨第十六类变应原的克隆表达、纯化及免疫原性鉴定

目的 获得大量具有良好IgE结合活性的粉尘螨第十六类变应原（Der f16）的重组变应原,以促进粉尘螨变态反应性疾病的特异性诊断及治疗的研究.方法 挑取经纯培养的粉尘螨,提取总RNA,根据已知Der f16基因序列设计引物,经RT-PCR扩增Der f16基因片段,产物连入pMD32-T载体中.扩增后,利用限制性内切酶EcoR Ⅰ和XhoⅠ双酶切将目的 基因片段连接到pET32a表达载体上,转化到大肠杆菌（E.coli BL21）中经IPTG诱导表达.表达载体经亲和层析纯化,SDS-PAGE检测蛋白纯度,Western bolt检测变应原免疫学活性.结果 以粉尘螨总RNA为模板成功克隆出Der f16基因,与数据库中Der f16基因同源性为100%;经IPTG诱导后,大肠杆菌大量表达Der f16蛋白,所获得的重组蛋白分子质量为73 ku,上清及沉淀物均有蛋白表达,且上清表达量高于沉淀物.重组Der f16能够与螨过敏患者血清中的IgE 反应,而不与健康者血清中的IgE反应.结论成功构建了Der f16的原核表达载体,并高效表达和纯化出具有免疫原性的Der f16重组蛋白.     

反枝苋花粉泛过敏原profilin基因的克隆表达、纯化及免疫学鉴定

目的克隆表达反枝苋花粉中泛变应原肌动蛋白抑制蛋白（profilin）,并鉴定其免疫学活性。方法利用RT-PCR结合RACE技术克隆反枝苋花粉泛变应原profilin的全长基因,并进行序列分析。设计带有酶切位点的特异性引物,采用RT-PCR获得整个反枝苋花粉profilin的开放阅读框,将其与pET28a载体连接并转化大肠杆菌BL21（DE3）进行诱导表达,Ni2＋亲和层析柱纯化重组蛋白,采用Western-blot检测其IgE结合活性。结果 cDNA核苷酸测序表明反枝苋花粉profilin的全长基因由675个碱基组成,开放阅读框为399 bp,编码131个氨基酸。重组反枝苋花粉profilin在大肠杆菌中可溶性表达,进一步经Western-blot检测具有良好的IgE结合活性。结论成功地克隆和表达了反枝苋花粉profilin,并具有良好的IgE应答免疫活性。     

重叠延伸聚合酶链反应克隆大鼠Foxo3a基因及序列测定

目的获得大鼠Foxo3a基因全长编码区基因。方法运用重叠延伸PCR方法从大鼠脾脏总RNA中扩增Foxo3a编码区基因全长,将其插入pMD19-T载体中进行酶切及测序鉴定。结果Foxo3a编码区基因全长2019bp,与GenBank中大鼠Foxo3a基因同源性为99．9％。结论运用重叠延伸PCR技术可成功克隆高GC含量大鼠Foxo3a的cDNA,为进一步研究该基因奠定了基础。     

铜绿假单胞菌mexA基因原核表达载体的构建

目的构建mexA基因的原核载体,为进一步表达MexA蛋白奠定基础。方法从临床分离多重耐药的铜绿假单胞菌抽提DNA,经PCR扩增出mexA1基因与PUC18克隆载体连接,PCR、酶切及测序鉴定,再以PUC/mexA1质粒为模板PCR扩增mexA目的基因,克隆至质粒pQE30中,构建表达质粒pQE30/mexA,构建的表达质粒pQE30/mexA转化大肠杆菌M15,培养后提取纯化重组质粒pQE30/mexA酶切鉴定。结果获得长约1.5 kbPCR mexA1产物,酶切结果显示所构建的重组质粒PUC/mexA1已成功地克隆了mexA1（1.5 kb）基因,序列分析结果与PAO1/mexA1序列相同,构建的表达质粒pQE30/mexA酶切鉴定,与插入pQE30的目的基因mexA（1.2 kb）片段相符。结论含mexA（1.2 kb）基因的原核表达载体构建成功,为进一步表达MexA蛋白奠定了基础。     

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.     

基于MORPAS的敖汉旗林家地金多金属成矿远景区预测

随着GIS技术实现多源地学信息的集成、分析以及融合,使得对于成矿远景区的定量化预测成为可能。本文在系统总结内蒙古敖汉旗林家地地区的地质条件以及区域成矿规律的基础上,利用GIS空间分析、主成分分析、聚类分析以及C-A分形理论等提取了包括地质、地球物理异常、地球化探异常以及遥感蚀变异常等在内的13个证据因子,并基于MORPAS平台应用证据权法成功建立了内蒙古敖汉旗林家地地区综合信息找矿模型,在此基础上成功圈定了15个I级成矿远景区和17个II级成矿远景区,同时对各成矿远景区结合实际地质情况进行了分析和评价。结果表明:内蒙古敖汉旗林家地地区已知金矿点大部分都落入圈定成矿远景区内,证明了该方法的合理性和可靠性。预测结果可为该区域的下一步找矿工作提供一定的技术支持和科学依据。     

Modeling Anisotropic Behavior of Gravelly Layer in Hualien,Taiwan

The Hualien Large-Scale Seismic Test (HLSST) program is under way to investigate soil-structure interaction during earthquakes in Hualien, a high-seismicity region in Taiwan. Since anisotropic characteristics of the gravelly layer affect the results of earthquake observation and forced vibration tests, the gravelly layer was modeled by an orthotropic elastic body in this paper to enhance the accuracy of the dynamic simulation analysis. The following conclusions are drawn. (1) The gravelly layer was modeled by an orthotropic elastic body whose model parameters were partly determined using results of earthquake observation. (2) The azimuth dependency of the shear wave velocity measured by the cross hole velocity logging can be expressed by the orthotropic elastic body model. (3) It can be explained by the orthotropic elastic body model that values of shear wave velocity measured by down-hole velocity logging were divided into two groups. (4) The initial shear moduli of undisturbed samples from the gravelly layer evaluated by cyclic triaxial tests agree with the calculated results by the orthotropic elastic body. (5) The anisotropic behavior of the model building during forced vibration tests can be expressed successfully by the orthotropic elastic body.     

Automatisierte zerstörungsfreie Zustandsuntersuchungen von Brückenbauwerken

Automated Non‐Destructive Assessment of Bridges Non‐destructive test methods in civil engineering are increasingly used for large‐area investigations of structures. The test methods ultrasonic‐echo, impact‐echo and radar are well suited for detailed investigations of pre‐stressed areas at pt‐concrete structures. A significant improvement of the results and their interpretations has been achieved in recent years using advanced data analysis procedures such as 3D‐reconstructions and the fusion of data. The automated application of the methods has been carried out successfully at decks and webs of pre‐stressed box girder bridges. The achieved results are discussed in detail. The measurement uncertainty and its importance for the results are presented considering as example a foundation slab.     

N型CaMnO3热电材料的制备及性能研究

该文通过实验研究了采用自燃法并在不同的温度下煅烧试样合成 Ca1-xSmxMnO3（x=0-0.06）粉末的工艺,以及在Ca位上掺杂不同含量的 Sm元素对材料性能的影响。自燃法结合球磨工艺可以制得平均粒径在2μm左右的Ca1-xSmxMnO3（x=0-0.06）超细粉末。X射线衍射（XRD）分析表明自燃法可以合成物相单一的 Ca1-xSmx MnO3（x=0-0.06）粉末材料。在CaMnO3热电材料的Ca位上掺杂 Sm 元素降低了材料的 Seebeck 系数,但同时也降低了材料的电阻率,而电导率增加幅度高于 Seebeck系数的降低幅度,故而总体上Ca1-xSmxMnO3（x=0-0.06）热电陶瓷材料的功率因子也增大了,从而有望使材料达到较高的热电优值,具有一定的商业应用价值。     

Determination of estrogens and estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast assay

A scheme of bioassay-directed analysis has been developed which combines a yeast assay screening for estrogenic activity with a liquid chromatographic-mass spectrometric (LC-MS/MS) chemical analysis, chromatographic fractionation, solid phase extraction and freeze-drying. The test scheme was applied on effluent samples collected from a municipal sewage treatment plant. The aim was to determine the substances responsible for main portion of the estrogenic activity in the samples and to compare the efficiency of different procedures for isolation and concentration of estogenicity. LC-MS/MS analyses were used for the quantification of 17beta-estradiol, estrone, estriol and 17alpha-ethinylestradiol, and the measured concentrations compared with the activities found in the yeast assay. Following conversion of the concentrations measured by LC-MS/MS to 17beta-estradiol equivalents it was concluded that freeze-drying, solid phase extraction and the chemical analysis gave comparable activities. Since estrone was the major estrogen in the effluent, this estrogen was also the major contributor to the estrogenic activity in the effluent. The estrogenic activity was equivalent to 4-7 ng/L of 17beta-estradiol. The yeast assay results from the tests of the chromatographic fractions showed that the major activity resides in the fraction where estrone, 17beta-estradiol and 17alpha-ethinylestradiol eluted. The activity of this fraction was substantially higher than the activity of the original wastewater sample. The reason for this could in part be explained by an inhibition of activity occurring in the original water sample.